Optical imaging is an advanced technique to image biotechnology products like gels, sensors, and micro arrays. It is also used increasingly to monitor specific molecular pathways in animals. This latter technique uses fluorescent dyes that absorb and emit near-infrared (NIR) radiation (700-900 nm) a spectral window where hemoglobin and water absorb minimally and therefore allow photons to penetrate several centimeters through tissue. Cyanine based NIR dyes (Cy7) are the popular ones among the very few commercially available fluorescent dyes showing NIR photophysical features. Unfortunately they are highly susceptible to degradation under biological conditions, and also known to undergo photochemical isomerization leading to nonfluorescent products. This proposal builds on our recent discovery that squaraine rotaxanes (SRs) are among the world's brightest and most stable fluorescent dyes. Current generation of SR dyes show absorption and emission profiles close to the Cy5 dyes. Herein we are proposing the synthesis of new generation NIR SR dyes which can be a superior substitute for Cy7 dyes. Our plan is to develop NIR absorbing bright and stable squaraine rotaxane dyes which can readily undergo conjugation with various biomolecules. They are likely to become superior replacements for the popular cyanine (Cy7) dyes and become extremely useful probes for both in vitro and in vivo imaging applications. The overall workplan for Phase I is to prepare novel bright and stable NIR-SR dyes and compare the properties of various bioconjugates that have been labeled with NIR-SR or Cy7. The two Specific Aims of the present proposal are: 1) SA1 to be performed at MTTI - Synthesize novel bright and stable NIR SR NHS esters and conjugate to 3 different types of biomolecules, a zinc coordinated dipicolylamine ligand (Zn-DPA), an antibody and a protein for comparison with Cy7 analogues. The first goal is to produce bright and stable NIR SR dyes and identifying the best two based on the chemical stability and brightness. This will be followed by conjugation of the squaraine rotaxane probes as well as Cy7 as a control probe to Zn-DPA, IgG protein and streptavidin . 2) SA2 to be perfomed at UND - In vitro evaluation of bioconjugates. In vitro molecular recognition performance of the NIR-SR and Cy7 fluorescent IgG and streptavidin bioconjugates developed at MTTI will be characterized using standard gel electrophoresis and streptavidin/biotin protocols. NIR-SR- and Cy7-Zn-DPA probes will be investigated for their interaction with S aureus and E coli bacteria membranes and their photostability using fluorescence microscopy. The goal is to demonstrate the advantage of using molecular probes labeled with an NIR-SR dye in 3 different systems compared with Cy7 dye. PUBLIC HEALTH RELEVANCE: The fluorescent probe developed by this research will be used in biotechnology as a stain for in vitro diagnostic devices, and in medical science as an in vivo probe to image disease in living animals. This optical imaging technology will help researchers discover new therapies and eventually it will help physicians choose the most appropriate therapy for a specific patient. This latter application is an example of how optical imaging will contribute to the evolving concept of personalized medicine.